This disclosure pertains to oligonucleotide-quencher conjugates with improved fluorescence characteristics, and to reagents suitable for incorporating novel quencher moieties into oligonucleotides. The disclosure also pertains to the use of oligonucleotide-quencher conjugates in detection methods for nucleic acid targets.
Nucleic acid hybridization probes are used for detection and discrimination of closely related nucleic acid targets. Fluorescence is often used to signal the hybridization event. Numerous types of fluorescence-based assays have emerged over past two decades as reviewed (Knemeyer and Marme, 2007). The so-called TaqMan probes (Heid et al., 1996). and Molecular Beacons (Tyagi et al., 1996) are the two most prominent examples of such methods. Both of these examples utilize both a fluorophore and a non-fluorescent quencher for signal generation with the quencher responsible for the probe's low background fluorescence in the unhybridized state.
The inherent specificity of natural DNA probes is not always sufficient to meet assay goals. Several methods for improved mismatch discrimination have been suggested and include use of a secondary structure (Bonnet et al., 1999), competitive hybridization (Morrison et al., 1998), solution-phase detection of polynucleotides using interacting labels and competitive hybridization, rigid nucleic acid backbones (Tolstrup et al., 2003), and Zip DNA (Paris et al., 2010). These and some other approaches have been discussed (Demidov and Frank-Kamenetskii, 2004). It has been also shown that stabilization of DNA duplexes by minor groove binding (MGB) agents allows for much shorter probes to be used at a given assay temperature and that such probes are more sensitive to single base variations (Kutyavin et al., 2000).
In addition to high hybridization specificity, ideal fluorescence-based nucleic acid probes should possess low fluorescence background, high signal and signal-to-background ratio (S/B). The Molecular Beacons can achieve high signals and S/B through the use of self-complementary ends that bring the fluorophore and the non-fluorescent DABCYL quencher in close proximity when probe is unhybridized and are far apart when the probe is bound to its target (Tyagi S. et al., 1998). Since the introduction of DABCYL several structural types of organic non-fluorescent quenchers have been described and patented. The most extensively explored azo dye-based quenchers are Eclipse Dark Quencher (U.S. Pat. No. 6,699,975), Diarylazo quenchers (U.S. Pat. No. 6,790,945), Black Hole Quenchers (U.S. Pat. No. 7,019,129), ZEN quencher (U.S. Pat. No. 7,439,341), BlackBerry Quenchers (U.S. Pat. No. 7,879,986) and Multi-Path Quenchers (Crisalli and Kool, 2011). Other known quenchers are based on rhodamine (QSY quenchers), rhodole (U.S. Pat. No. 6,399,392), triarylmethane, fluorescein (U.S. Pat. No. 6,323,337), anthraquinone based Iowa Black quencher (U.S. Pat. No. 7,803,536) and nitro-substituted cyanine dyes (U.S. Pat. No. 7,166,715).
MGB-labeled hybridization probes called Pleiades (Lukhtanov et al., 2007, U.S. Pat. No. 7,381,818) offer the benefits of high signal and S/B without the need for the Molecular Beacon's self-complementary stem structure. The key structural features of the Pleiades probes are strategically positioned 5′-end MGB and fluorophore and 3′-end Eclipse Dark quencher. All three components work in a coordinated manner to yield a unique signal generation mechanism with low background fluorescence, high signal and S/B (Lukhtanov et al., 2007). In addition, the 5′-positioning of the MGB prevents the probe from being cleaved by Taq polymerase. The Eclipse Dark quencher (U.S. Pat. No. 6,699,975) used in these probes has been designed to optimize fluorescence quenching based on the fluorescence resonance energy transfer (FRET), which requires an overlap of the absorption spectrum of the quencher with the emission spectrum of the fluorophore. The absorption spectrum of the Eclipse Dark quencher overlaps efficiently with most common fluorophore (Fluorescein, tetramethylrhodamine, Texas Red). When used under PCR-relevant conditions (55-70° C.) the MGB-probes are 15-20 bases long. At this length hybridization of the probe to a target provides sufficient spatial separation between the fluorophore and the quencher thus eliminating most of the FRET quenching.
Despite the recognized advantages of using FRET-based Pleiades probes, there are certain situations when the FRET mechanism is detrimental to probe's performance. For example, very short MGB-probes (8-12 bases long) are ideal for low temperature (20-50° C.) applications if high hybridization specificity is required. For those probes, however, hybridization to a target does not provides sufficient spatial separation between the fluorophore and the quencher, leading to significant residual FRET quenching and consequently to a low fluorescence signal and reduced sensitivity. To fully employ the mismatch discrimination advantages of short fluorogenic MGB-probes it is, therefore, necessary to optimize the quenching mechanism with the goal of increasing fluorescence signal.
One possible solution is based on the use of so-called “non-FRET” quenchers. Contact quenching (also known as quenching by “touching” or collisional quenching) is not based on the long range (20-60 Å) FRET mechanism and requires close contact between fluorophore and quencher (U.S. Pat. No. 6,150,097). The described method works especially well in the Molecular Beacons wherein the “touching” is enhanced by the formation of the self-complementary double-stranded stem. Linear probes, however, do not demonstrate very efficient quenching. Based on dynamics of fluorophore-quencher interaction, quenching was categorized as being either dynamic or static in complex formation (Lakowicz, 2007). Three different contact quenching mechanisms were identified: intersystem crossing, electron exchange, and photoinduced electron transfer (PET). At least the last two mechanisms have been shown to be present in known nucleic acid probes. For example, the electron exchange (also known as Dexter interaction) is present (along with FRET) in linear dual labeled probes (such as TaqMan) and requires temporary orbital overlap. The photoinduced electron transfer between a fluorescent dye and a guanine base is the quenching mechanism for the so-called Smart probes (U.S. Pat. No. 7,262,007) or for probes with a guanine base in close proximity of the dye (U.S. Pat. No. 6,699,661). Examples of possible PET-mediated quenching by a methanesulfonylaminoindole (U.S. Pat. No. 7,759,470) or by nitroindole nucleosides (EP Patent No. EP1384789) are described. Lukhtanov et al. 2007 demonstrated that the CDPI3-type MGB-dependent quenching invokes the PET mechanism as well. An example of the static quenching is described by Johansson et al., 2002. It is characterized by a formation of a ground state complex (hetero-dimer) between a fluorophore and a Black Hole quencher accompanied by a significant change in absorption spectra of the dyes. FIG. 1 shows a general schematic diagram for some of these quenching mechanisms in oligonucleotide probes containing MGB.
Published studies suggest that at least some of the existing quenchers already possess the required contact quenching properties and all is needed is to somehow reduce or eliminate the accompanied FRET quenching. However, problematically, all popular quenchers of common fluorophores (500-600 nm emission range) that presumably employ some degree of contact quenching also possess a significant FRET component. Moreover, those quenchers are typically designed to maximize the FRET effect.
Due to the described deficiencies, there is a need for redesign of the quenchers with the goal of weakening the FRET effect while preserving the contact quenching.